Diagnostic and examination method for cancer of the colon using tannase as indication

ABSTRACT

A diagnostic agent for colon cancer, which comprises a reagent for detecting a tannase high-producing bacterium or measuring an amount of tannase contained in an intracolonic microflora sample, and a test method for colon cancer, which comprises the step of detecting a tannase high-producing bacterium or measuring an amount of tannase contained in an intracolonic microflora sample.

TECHNICAL FIELD

[0001] The present invention relates to a diagnostic agent for coloncancer and a test method for colon cancer.

BACKGROUND ART

[0002] In recent years, morbidity of colon cancer is increasing with thewesternization of diets in Japan. It is said that high-fat diets andlow-fiber diets change the intestinal microflora and increase productionof carcinogens, and further, reduction of feces amount prolongsintestinal feces retention time and hence prolongs contact time betweencarcinogens and the intestinal canal, resulting in increase of risks ofoncogenesis. Bacteria in the intestine extremely closely relate tohealth and diseases of hosts and considered to associate with coloncancer through diet components and body components.

[0003]Streptococcus (St.) bovis is known as a pathogenic bacterium ofinfectious endocarditis, and high concurrent incidence of infectiousendocarditis due to this bacterium and colon cancer attracts attentionin Europe and United States (Honberg P. Z. et al., Lancet, i: 163-164,1987). Meanwhile, Osawa et al. isolated a bacterium having a tannaseactivity, which hydrolyzes an ester bond in tannic acid to releasegallic acid, from feces of koala eating eucalyptus containing tannin ata high concentration, and identified this bacterium as St. bovis biotypeI (Osawa R. et al., Appl. Environ. Microbiol., 56: 829-831, 1990). Thisbacterium has the tannase activity for degrading tannin to releasegallic acid, and contains decarboxylase that decarboxylates gallic acidinto pyrogallol, and it is proposed that it should be newly designatedas St. gallolyticus. Osawa inferred a possibility that this St.gallolyticus was a bacterium identical to St. bovis isolated from acolon cancer patient concurrently having infectious endocarditis (OsawaR., RIKEN Symposium Abstracts, “Classification and Ecology of LacticAcid Bacteria”, pp. 36-45, 1996).

[0004] In general, a substance referred to as tannin belongs topolyphenols, natural substances constituting an important portion ofphenolic compounds, and has toxic and growth-inhibitory actions onmicroorganisms as well as an astringent taste. While variousclassifications of tannin have been proposed, tannin is largelyclassified into hydrolysable tannin and condensed tannin forconvenience. The former is structurally pyrogallol tannin, and it isalso referred to as pathological tannin since it is contained in nutgallor gall nut in a large amount. On the other hand, the latter is catecholtannin, and it is also referred to as physiological tannin since it is anormal component of plants. The hydrolysable tannin has a chemicalstructure in which phenolic acid (gallic acid, ellagic acid etc.) bindsto a saccharide as a core through an ester bond. It is known that thishydrolysable tannin is hydrolyzed by tannase, which is atannin-degrading enzyme mainly produced by fungi such as Aspergillus andCandida living in soil. However, production of tannase by bacterialiving in the intestinal canal of animals had not been reported beforethe publication of Osawa.

[0005] Since then, it has been reported that Lonepinella koalarum wasisolated from feces of koala as a tannase-positive bacterium, inaddition to St. gallolyticus, and also isolated mainly from herbivorousanimals (Osawa R., Syst. Appl. Microbiol., 15: 144-147, 1992). Recently,it has been further reported that Lactobacillus (L.) plantarum wasisolated from human feces. L. plantarum is a lactobacillus mainlyisolated from silos, and infectious endocarditis due to L. plantarum hasalso been reported (Oakey H. J. et al., J. Appl. Bacteriol., 78:142-148, 1995).

[0006] The onset mechanism of colon cancer has been studied by manyresearchers, and existence of active oxygen can be mentioned as a partof the mechanism (Babbs C. F. et al., Free Rad. Biol & Med., 8: 191-200,1990). Active oxygen exhibits a sterilizing action againstmicroorganisms entering into a living body to protect the body frominfection. On the other hand, active oxygen has a risk of increasingadverse reaction products that can damage body functions by radicalchain reactions and thus worsening various pathological conditions. Itis considered that balance of production and elimination of activeoxygen is lost in, in particular, aging and life habit diseases such asarteriosclerosis and cancer, leading to worsening of conditions over along time.

[0007] Many chemical substances have been discovered as substancesproducing active oxygen, and gallic acid and pyrogallol are among them(Khan N. S., Mutagenesis, 13: 271-274, 1998).

DISCLOSURE OF THE INVENTION

[0008] The inventors of the present invention established a hypothesisabout the relationship between bacteria having a tannase activity andcolon cancer as follows. When tannase acts on hydrolysable tannin,gallic acid is produced, pyrogallol is further produced in the presenceof gallic acid decarboxylase, and thus a chemical substance producingactive oxygen is released. If a tannase-positive bacterium is involvedin this pathway, active oxygen is produced in the presence of thetannase-positive bacterium, and this would be one of causes of onset ofcancer, in particular, colon cancer.

[0009] Under such a background, the inventors of the present inventionassiduously studied in order to elucidate how a bacterium having atannase activity in the colon associates with colon cancer. As a result,they found that Staphylococcus (S.) lugdunensis, a tannase-positivebacterium, was found only in colon cancer patients, and, further foundthat S. lugdunensis produced a larger amount of tannase in comparisonwith other tannase-positive bacteria. Accordingly, it is considered thata large amount of tannase produced by S. lugdunensis causes coloncancer, and that a part of colon cancer cases can be diagnosed or testedby detecting a tannase high-producing bacterium or tannase.

[0010] The present invention was accomplished based on theaforementioned findings and provides the following.

[0011] (1) A diagnostic agent for colon cancer, which comprises areagent for detecting a tannase high-producing bacterium contained in anintracolonic microflora sample.

[0012] (2) A diagnostic agent for colon cancer, which comprises areagent for detecting Staphylococcus lugdunensis contained in anintracolonic microflora sample.

[0013] (3) A test method for colon cancer, which comprises the step ofdetecting a tannase high-producing bacterium contained in anintracolonic microflora sample.

[0014] (4) A test method for colon cancer, which comprises the step ofdetecting Staphylococcus lugdunensis contained in an intracolonicmicroflora sample.

[0015] (5) A diagnostic agent for colon cancer, which comprises areagent for measuring an amount of tannase contained in an intracolonicmicroflora sample.

[0016] (6) The diagnostic agent according to (5), wherein the amount oftannase is measured based on an enzymatic activity.

[0017] (7) The diagnostic agent according to (5), wherein the amount oftannase is measured by an immunoassay.

[0018] (8) A test method for colon cancer, which comprise the step ofmeasuring an amount of tannase contained in an intracolonic microflorasample.

[0019] (9) The method according to (8), wherein the amount of tannase ismeasured based on an enzymatic activity.

[0020] (10) The method according to (8), wherein the amount of tannaseis measured by an immunoassay.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIG. 1 shows quantification of gallic acid, pyrogallol andphloroglucinol by HPLC.

[0022]FIG. 2 shows comparison of amounts of tannase produced by St.bovis biotype I, L. plantarum and S. lugdunensis.

BEST MODE FOR CARRYING OUT THE INVENTION

[0023] Hereafter, embodiments of the present invention will be explainedin detail.

[0024] The present invention provides a diagnostic agent for coloncancer comprising a reagent for detecting a tannase high-producingbacterium contained in an intracolonic microflora sample and a testmethod for colon cancer comprising the step of detecting a tannasehigh-producing bacterium contained in an intracolonic microflora sample.The present invention also provides a diagnostic agent for colon cancercomprising a reagent for detecting S. lugdunensis contained in anintracolonic microflora sample and a test method for colon cancercomprising the step of detecting S. lugdunensis contained in anintracolonic microflora sample.

[0025] In the present invention, the term “tannase high-producingbacterium” refers to a bacterium producing 0.3 mM or more, preferably0.5 mM or more, of gallic acid and pyrogallol as a total amount of themas measured by the tannase activity measurement method using HPLCdescribed later in Example 2.

[0026] The tannase high-producing bacterium is preferably S.lugdunensis.

[0027]S. lugdunensis has a high tannase producing ability and generallysatisfies the aforementioned definition of the tannase high-producingbacterium. Hereinafter, for convenience, S. lugdunensis and othertannase high-producing bacteria are generically referred to as a tannasehigh-producing bacterium.

[0028] The expression “intracolonic microflora sample” means a samplecontaining bacterial flora in the colon, and examples thereof includefeces, intestinal canal wash and so forth.

[0029] The tannase high-producing bacterium contained in an intracolonicmicroflora sample can be detected by culturing a bacterium existing inthe intracolonic microflora sample for isolation and measuring thetannase producing ability of the bacterium, or identifying the bacteriumin the intracolonic microflora sample to determine whether it is atannase high-producing bacterium or not. In the culture for isolation,the tannase-positive bacterium is preferably isolated by using atannin-treated agar medium. Further, identification of the bacterium ispreferably performed after the isolation of the tannase-positivebacterium, because such a procedure facilitates the identification. Thebacterium can be identified by searching bacteriological properties suchas biochemical properties and/or searching homologies of nucleotidesequences that can be used for identification of bacteria. Examples ofthe nucleotide sequences that can be used for identification of bacteriainclude a nucleotide sequence of the 16S-rRNA gene.

[0030] Hereafter, the detection method will be specifically explained byusing S. lugdunensis as an example.

[0031]S. lugdunensis can be detected by isolating a tannase-positivebacterium from an intracolonic microflora sample by using atannin-treated agar medium, searching various already known biochemicalproperties for the isolated tannase-positive bacterium, or searchinghomology of the nucleotide sequence of the 16S-rRNA gene.

[0032] Examples of the biochemical properties include various propertiessuch as degrading abilities for glucose, fructose, D-mannose, maltose,lactose, D-trehalose, D-mannose, xylitol, D-melibiose, D-raffinose,D-xylose, sucrose, α-methyl-D-glucoside and N-acetyl-D-glucosamine,ability of reducing nitrate to nitrite, alkaline phosphatase-producingability, acetylmethylcarbinol-producing ability, argininehydrase-producing ability, and urease-producing ability, and thebiochemical properties can be tested by using these as indexes.

[0033] The reagent for detecting a tannase high-producing bacteriumcontained in the diagnostic agent of the present invention is at leastone of reagents used for the aforementioned detection of tannasehigh-producing bacterium and can be suitably selected by those skilledin the art depending on the detection method. Specific examples includea tannin-treated agar medium, bacterium identification kit, primers usedfor determination of nucleotide sequences by PCR and so forth. Thediagnostic agent according to this embodiment can be produced by atechnique selected from those usually used for the production of adiagnostic agent depending on the reagent for detecting a tannasehigh-producing bacterium. When the reagent for detecting a tannasehigh-producing bacterium is composed of multiple reagents, thesereagents may constitute a kit. The reagent for detecting a tannasehigh-producing bacterium may be used as a composition in combinationwith a carrier that is acceptable for the use in diagnostic agents.

[0034] Components of the diagnostic agent according to this embodimentmay include, for example, Brain-Heart Infusion Agar (BHI agar) treatedwith tannic acid as a selective medium and a bacterium identificationkit.

[0035] Existence of a tannase high-producing bacterium can be aneffective index for the diagnosis or test of colon cancer. That is,presence or absence of colon cancer can be determined based on thedetection result for tannase high-producing bacteria in combination withother test results as required. The reason why the existence of thetannase high-producing bacterium can be an effective index for thediagnosis or test of colon cancer is considered that, as shown in theExamples described later, the existence of tannase in a large amountcauses colon cancer. Therefore, it is considered that the amount oftannase in the colon can also be an effective index for the diagnosis ortest of colon cancer. That is, presence or absence of colon cancer canbe determined based on the measurement result for tannase in combinationwith other test results as required.

[0036] Therefore, the present invention also provides a diagnostic agentfor colon cancer comprising a reagent for measuring an amount of tannasecontained in an intracolonic microflora sample and a test method forcolon cancer comprising the step of measuring an amount of tannase in anintracolonic microflora sample.

[0037] The amount of tannase may be measured by measuring the enzymaticactivity of tannase, or measuring tannase itself by an immunoassay.

[0038] The measurement of tannase amount based on the enzymatic activitycan be performed by allowing methyl gallate, which is a substrate oftannase, to react with a sample and measuring the amount of theenzymatic reaction product using HPLC or a color development method. Forexample, the measurement is performed by using methyl gallate as asubstrate and quantifying gallic acid as the degrading product by HPLC;mixing methyl gallate and a test specimen and measuring the absorbanceat 440 nm; or mixing methyl gallate and a test specimen and observingwhether the solution turns to green and then brown color. Thequantification is preferably performed based on absorbance.

[0039] The measurement of tannase amount by an immunoassay can beperformed by a usual immunoassay using an antibody directed to tannase.

[0040] Hereafter, an example of a method for producing antibodiesdirected to tannase and a method for measuring tannase will beexplained.

[0041] 1) Purification of Tannase

[0042] Tannase can be purified from a supernatant obtained by culturinga tannase-positive bacterium, disrupting cultured cells by, for example,ultrasonication, and centrifuging the culture. The purification can beperformed by a suitable combination of electrophoresis and variouschromatography methods (ion exchange chromatography, hydrophobicchromatography, gel filtration etc.) using a tannase activity as anindex. The tannase activity can be measured as described above.

[0043] 2) Production of Antibody

[0044] Polyclonal antibodies directed to tannase can be obtained by, forexample, immunizing a rabbit or the like with purified tannase. Further,monoclonal antibodies can be obtained by immunizing a mouse or the likeand fusing its spleen cell with a myeloma cell.

[0045] 3) Method for Measuring Tannase in Human Feces

[0046] Colon cancer can be diagnosed or tested by measuring tannasecontained in an intracolonic microflora sample, for example, feces byimmunological techniques using the produced antibodies directed totannase (anti-tannase antibodies). As the immunoassay, known methods canbe employed, and examples thereof include enzyme immunoassay,radioimmunoassay, chemiluminescence immunoassay,electrochemiluminescence immunoassay, immunochromatography, Westernblotting, passive particle agglutination, passive hemagglutination,latex particle agglutination and so forth.

[0047] An exemplary procedure of the immunoassay is as follows.Anti-tannase antibodies immobilized on a carrier such as a microtiterplate as a solid phase are allowed to react with a specimen sample and,after washing, allowed to react with anti-tannase antibodies labeledwith a labeling substance as second antibodies. After washing, thelabeling substance is quantified.

[0048] As the carrier for immobilizing anti-tannase antibodies on asolid phase, besides the microtiter plate, any of carriers such asmagnetosensitive beads, plastic beads, nitrocellulose membrane, nylonmembrane, erythrocytes, gelatin particles, polyamino acid particles andlatex particles can be used.

[0049] In the case of the enzyme immunoassay, an enzyme such asperoxidase, alkaline phosphatase and β-galactosidase is used as thelabeling substance of the second antibodies, and the labeling substanceis quantified by adding an enzyme substrate and measuring colordevelopment of the substrate using a spectrophotometer.

[0050] As the labeling substance of the second antibody, besidesenzymes, any quantifiable substances such as fluorescent substances,radioactive substances, bioluminescent or chemiluminescent substances,electrochemiluminescent substances, dyes and metals can be used.

[0051] In the measurement of the tannase amount in the presentinvention, absolute values do not necessarily need to be obtained, butit is sufficient to determine whether the values exceed a predeterminedvalue. The predetermined value is a value at which colon cancer issignificantly observed.

[0052] The reagent for measuring the amount of tannase, which isincluded in the diagnostic agent of the present invention is atleast-one of reagents used for the aforementioned measurement of theamount of tannase and can be suitably selected by those skilled in theart depending on the measurement method. Specific examples thereofinclude tannase substrates, anti-tannase antibodies and so forth. Thediagnostic agent according to this embodiment can be produced by atechnique selected from those usually used for the production of adiagnostic agent depending on the reagent for measuring the amount oftannase. When the reagent for measuring the amount of tannase iscomposed of multiple regents, these reagents may constitute a kit. Thereagent for measuring the amount of tannase may be used as a compositionin combination with a carrier that is acceptable for the use indiagnostic agents.

[0053] Components of the diagnostic agent according to this embodimentmay include, in the case of an enzyme immunoassay kit, for example, aplate as a solid phase on which anti-tannase monoclonal antibody isimmobilized, enzyme-labeled anti-tannase monoclonal antibody, tannasestandard, a specimen-diluting buffer, a washing solution and areaction-terminating solution.

EXAMPLES

[0054] The present invention will be explained more specifically withreference to the following examples. However, the scope of the presentinvention is not limited to these examples.

Example 1 Isolation of Tannase-Positive Bacterium from Human Feces

[0055] 1) Collection of Clinical Specimen

[0056] Feces collected from 167 in total of patients and healthysubjects who underwent colon endoscopy at Tokyo Medial UniversityHachioji Medical Center from April, 1999 were used as specimens.Patients in whom colon endoscopy revealed diseases besides colon canceror polyp of colon in the lower digestive tract were excluded. For coloncancer patients from whom a tannase-positive bacterium was detected,specimens were collected before and after the colon cancer operation.The specimens collected after the operation was collected two weeksafter the operation in consideration of influence of antibiotics usedduring and after the operation.

[0057] Each of collected clinical specimens (feces) was inoculated in aBrain-Heart Infusion (BHI) liquid medium (Difco) to which aStreptococcus selective supplement (Oxoid) was added, and anaerobicallycultured overnight at 37° C. The Streptococcus selective supplement wasadded in an amount of 0.4 ml per 100 ml of the BHI liquid medium.

[0058] 2) Isolation of Bacterium Having Tannase Activity

[0059] Culture for isolation of a bacterium having a tannase activitywas performed as follows according to the method of Osawa et al. (OsawaR. et al., Appl. Environ. Microbiol., 56: 829-830, 1990). AStreptococcus selective supplement-added tannin-treated BHI agar mediumwas prepared by the following method.

[0060] A BHI agar medium (Oxoid) was autoclaved at 121° C. for 15minutes, and the Streptococcus selective supplement was added to it toprepare a plate agar medium. It was left overnight at 37° C.Subsequently, a PBS buffer to which tannic acid (Kanto Kagaku) was addedat 2% (w/v) and sterilized by filtration, was poured over the surface ofthe plate agar medium left overnight at 37° C. so as to be spreadextremely thinly and uniformly and left standing for 20 minutes. Aftertannic acid and a protein in the medium formed a water-insoluble complexand the surface of the plate agar medium turned white, the tannic acidsolution on the surface was removed, and excess tannic acid on thesurface of the plate agar medium was washed away with a PBS buffer. Thewashing was repeated 3 times. After a liquid on the surface was removed,the plate agar medium was left for 1 hour in a substantially verticalstate, and then a liquid left at the bottom was removed. 100 μl ofclinical specimen cultured in 1) was spread on the preparedStreptococcus selective supplement-added tannin-treated BHI agar mediumand anaerobically cultured at 37° C. for 3 days. Colonies found to besurrounded by a clear zone were isolated as tannase-positive bacteria.

[0061] 3) Identification of Isolated Tannase-Positive Bacterium

[0062] The tannase-positive bacteria microscopically found to be cocciwere identified by using Staphylococcus and Micrococcus IdentificationKit API STAPH (bioMerieux Japan), those found to be streptococci wereidentified by using Streptococcus Identification Kit AP120 STREP(bioMerieux Japan), and those found to be bacilli were identified byusing Lactic acid bacterium identification kit API 50CHL (bioMerieuxJapan). Further, as for S. lugdunensis, the nucleotide sequence of the16S-rRNA gene was determined according to the method of Ezaki et al.(Ezaki T. et al., Int. J. Syst. Bacteriol., 44: 130-136, 1994) toconfirm homology.

[0063] 4) Results

[0064] Among the fecal specimens collected from the 167 subjects,tannase-positive bacteria were detected from the specimens of 30subjects. The details of these subjects were 11 (21.6%) of 51 coloncancer patients, 11 (20.4%) of 54 colon polyp patients and 8 (12.9%) of62 healthy subjects (Table 1). L. plantarum was detected in all groups,but S. lugdunensis was mostly detected from the colon cancer patients,but detected from only one colon polyp patient. Specimens were collectedfrom the colon cancer patients before and after the colon canceroperation, but no difference was noted between the detection resultsbefore and after the operation. TABLE 1 Isolation of tannase-positivebacteria from colon cancer patients, colon polyp patients and normalsubjects Positive Patients S. lugdunensis L. plantarum Negative TotalColon cancer 9 2 40 51 patients Colon polyp 1 10 43 54 patients Normal 08 54 62 subjects

[0065] The nucleotide sequence of the 16S-rRNA gene of isolated S.lugdunensis was determined (SEQ ID NO: 1). As a result of the homologysearch utilizing the database of the DNA Data Bank of JAPAN (DDBJ), thisnucleotide sequence showed homology of 99% with the registered 16S-rRNAgene sequence of S. lugdunensis.

Example 2 Measurement of Tannase Activity

[0066] 1) Measurement of Tannase Activity Using HPLC

[0067] As a substrate of tannase, methyl gallate (Sigma) having thebasic structure of tannic acid was used. Further, as the standarddegradation products generated by the degradation of methyl gallate bytannase, gallic acid (Sigma), pyrogallol (Sigma) and phloroglucinol(Sigma) were used. Each of these was prepared at a concentration of 5 mMin 33 mM NaH₂PO₄.

[0068] The tannase-positive bacteria shown in FIG. 2 were cultured in atannin-treated BHI agar medium under an anaerobic condition at 37° C.for 72 hours. Then, the bacteria were scraped off with a cotton swab andsuspended in 2.5 ml of a substrate solution to prepare a suspension ofMcFarland turbidity 4, and the suspension was incubated at 37° C. for 24hours. 1 ml of the incubated suspension was centrifuged at 3,000×g for 1minute. To 400 μl of the supernatant, an equivalent volume of 0.1 Mhydrochloric acid was added and the mixture was further subjected tocentrifugal filtration through a membrane filter to obtain a samplesolution. Further, as a control, the same pretreatment was performed byusing Escherichia (E.) coli.

[0069] The decomposition products generated by the decomposition ofmethyl gallate by tannase were separated and quantified by HPLC. Underthe conditions used in this example, retention times of gallic acid,pyrogallol and phloroglucinol were 5.54 minutes, 6.53 minutes and 7.20minutes, respectively (FIG. 1). Any peak of methyl gallate was notdetected.

[0070] The total amounts of gallic acid and pyrogallol, which weremetabolites produced by the bacteria used in this example from methylgallate as the substrate were shown in FIG. 2. Among the isolatedclinical strains of tannase-positive bacteria, St. bovis biotype I, L.plantarum and S. lugdunensis, it was S. lugdunensis that produced themetabolites in the largest amount. As for E. coli NIHJ JC-2, no peak wasdetected for gallic acid and pyrogallol. Any bacteria used in thisexample did not show a peak for phloroglucinol.

[0071] The result that S. lugdunensis produced the metabolites in thelargest amount among the tannase-positive bacteria and was detectedmainly from colon cancer patients indicated the association between thetannase activity and colon cancer. Therefore, it is considered that theexistence of tannase high-producing bacteria can be used as an index intests and diagnoses of colon cancer.

[0072] 2) Measurement of Tannase Activity by Color Development

[0073] Instead of the measurement by HPLC, the tannase activity was alsomeasured by a color development method (performed according to themethod of Osawa et al. (Osawa R. et al., Appl. Environ. Microbiol. 59:1251-1252, 1993)). Methyl gallate (10 mM) dissolved in a phosphatebuffer (33 mM NaH₂PO₄) and a test specimen were mixed at a ratio of 2:1and allowed to react at 37° C. for 60 minutes under an aerobiccondition. After the reaction, an equivalent volume of a saturatedNaHCO₃ solution was added to the reaction mixture, and the mixture wasleft standing at room temperature for 20 minutes, and the absorbance wasmeasured at 440 nm by using a spectrophotometer (DOUBLE-BEAMSPECTROPHOTOMETER UV-190: Shimadzu Corporation). At the same time, thereaction mixture was observed by visual inspection. When the solutionturned to green and then brown color, which indicates a high tannaseactivity, the result was determined to be positive. When the solutionwas colorless or light yellow, the result was determined to be negative.

Example 3 DNA Damage by Tannin Decomposition Product

[0074] After E. coli JM109 harboring pBR322 was cultured in an LB mediumwith shaking, plasmid DNA was extracted by the alkaline method, and theCsCl-ethidium bromide density gradient centrifugation was performed toprepare supercoiled plasmid DNA located in a lower layer.

[0075] 1 μl of supercoiled plasmid DNA, 1 μl of 1 M phosphate buffer (pH7.4) and test specimens (methyl gallate, gallic acid and pyrogallol) atconcentrations of 0, 0.5, 1, 2.5, 5, 10 and 20 mM were mixed andsupplemented with purified water to prepare a reaction mixture of atotal volume of 10 μl. Each reaction mixture was incubated at 37° C. for4 hours, and DNA of a supercoiled form and DNA of a nicked open circularform were separated by agarose gel electrophoresis to determine theactivity of the test specimens to damage DNA.

[0076] Almost no DNA-damaging ability was observed for methyl gallate,which is a substrate of tannase, but gallic acid and pyrogallolconcentration-dependently damaged DNA. Pyrogallol had stronger damagingability.

[0077] These results and the association between the tannasehigh-producing bacteria and colon cancer support the hypothesis thattannase in a large amount causes colon cancer. Therefore, it isconsidered that the tannase detection result can also be a useful indexin tests and diagnoses of colon cancer.

Example 4 Purification of Tannase

[0078] 1) Preparation of Crude Enzyme Solution

[0079]L. plantarum No. 67 was cultured in a tannin-treated BHI agarmedium under an anaerobic condition at 37° C. for 3 days (ANAEROBOX AZseries, Hirasawa Seisakusho). One colony was inoculated in 70 ml of BHImedium and cultured with stirring at 37° C. for 18 hours under ananaerobic condition. After the culture, 20 ml of the bacterial solutionwas added to 1,500 ml of BHI medium and cultured at 37° C. for 24 hourswith stirring under an anaerobic condition. Bacterial cells werecollected by centrifuging the culture broth (6,000 rpm, 10 minutes, 4°C.) and suspended in 2.5 ml of 10 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 50mM NaCl (hereinafter, referred to as M buffer). To the bacterialsuspension, 2.5>1 of N-acetylmuramidase SG (Seikagaku Corporation)prepared at 10 mg/ml by using the M buffer was added per ml of thesuspension, and the suspension was allowed to react at 37° C. for 30minutes under an aerobic condition. Further, the bacterial suspensionwas subjected to ultrasonication and centrifuged (6,000 rpm, 10 minutes,4° C.), and the centrifugation supernatant was collected to obtain acrude enzyme solution.

[0080] 2) Fractionation by Ion Exchange Chromatography

[0081] 25 ml of the crude enzyme solution was applied to DEAE-SepharoseFast Flow (Pharmacia) packed in Column XK 26/20 (2.6×20 cm: Pharmacia),and unadsorbed substances were eluted with 300 ml of 0.05 M Tris-HCl (pH7.0) at a flow rate of 5 ml/min. Then, proteins were eluted with aconcentration gradient of 0 to 0.4 M NaCl. Further, the remainingproteins were completely eluted with 0.4 M NaCl over 20 minutes. Afterthe elution of the unadsorbed substances, the sample was fractionatedinto 5-ml fractions. For each fraction, protein content, tannaseactivity and active oxygen were measured. Fractions showing the activitywere lyophilized by using a freeze dryer (FREEZVAC-1: Tozai Tsusho),dissolved in 0.05 M Tris-HCl (pH 7.0) and dialyzed against 0.05 MTris-HCl (pH 7.0).

[0082] 3) Fractionation by Gel Filtration Chromatography

[0083] 2 ml of the fraction having a tannase activity obtained by theion exchange chromatography was overlaid on Superose 12 (Pharmacia)packed in Column HR 16/50 (1.6×50 cm: Pharmacia). As an elutionsolution, 0.05 M NaCl, 0.05 M Tris-HCl (pH 7.0) was used. The flow ratewas 2 ml/min and the eluate was fractionated into 2-ml fractions. Afraction collector was started 13 minutes after the sample was overlaidon a column. For each fraction, protein content, tannase activity andactive oxygen were measured. Fractions showing the activity werecollected.

[0084] The sample at each purification step was analyzed by SDS-PAGE. Asa result, tannase was purified as almost a single band (45 kDa) afterthe purification using Superose 12.

INDUSTRIAL APPLICABILITY

[0085] According to the present invention, diagnosis of colon cancer isenabled based on detection of a tannase high-producing bacterium such asS. lugdunensis and/or detection of tannase.

1 1 1 1490 RNA Staphylococcus lugdunensis 1 uuagaguuug aucauggcucaggaugaacg cuggcggcgu gccuaauaca ugcaagucga 60 gcgaacagau aaggagcuugcuccuuugac guuagcggcg gacgggugag uaacacgugg 120 guaaccuacc uauaagacugggacaacuuc gggaaaccgg agcuaauacc ggauaauaug 180 uugaaccgca ugguucaauagugaaagaug guuuugcuau cacuuauaga uggacccgcg 240 ccguauuagc uaguuggugagguaacggcu caccaaggca acgauacgua gccgaccuga 300 gagggugauc ggccacacuggaacugagac acgguccaga cuccuacggg aggcagcagu 360 agggaaucuu ccgcaaugggcgaaagccug acggagcaac gccgcgugag ugaugaaggu 420 cuuaggaucg uaaaacucuguuauuaggga agaacaaacg uguaaguaac ugugcacguc 480 uugacgguac cuaaucagaaagccacggcu aacuacgugc cagcagccgc gguaauacgu 540 agguggcaag cguuauccggaauuauuggg cguaaagcgc gcguaggcgg uuuuuuaagu 600 cugaugugaa agcccacggcucaaccgugg agggucauug gaaacuggaa aacuugagug 660 cagaagagga aaguggaauuccauguguag cggugaaaug cgcagagaua uggaggaaca 720 ccaguggcga aggcgacuuucuggucugua acugacgcug augugcgaaa gcguggggau 780 caaacaggau uagauacccugguaguccac gccguaaacg augagugcua aguguuaggg 840 gguuuccgcc ccuuagugcugcagcuaacg cauuaagcac uccgccuggg gaguacgacc 900 gcaagguuga aacucaaaggaauugacggg gacccgcaca agcgguggag caugugguuu 960 aauucgaagc aacgcgaagaaccuuaccaa aucuugacau ccuuugaccg cucuagagau 1020 agagucuucc ccuucgggggacaaagugac agguggugca ugguugucgu cagcucgugu 1080 cgugagaugu uggguuaagucccgcaacga gcgcaacccu uaagcuuagu ugccaucauu 1140 uaguugggca cucuaaguugacugccggug acaaaccgga ggaagguggg gaugacguca 1200 aaucaucaug ccccuuaugauuugggcuac acacgugcua caauggacaa uacaaagggc 1260 agcgaaaccg cgaggucaagcaaaucccau aaaguuguuc ucaguucgga uuguagucug 1320 caacucgacu acaugaagcuggaaucgcua guaaucguag aucagcaugc uacggugaau 1380 acguucccgg gucuuguacacaccgcccgu cacaccacga gaguuuguaa cuacccgaag 1440 ccgguggagu aaccauucggagcuagccgu cgaaggugga cuaagauggu 1490

What is claimed is:
 1. A diagnostic agent for colon cancer, whichcomprises a reagent for detecting a tannase high-producing bacteriumcontained in an intracolonic microflora sample.
 2. A diagnostic agentfor colon cancer, which comprises a reagent for detecting Staphylococcuslugdunensis contained in an intracolonic microflora sample.
 3. A testmethod for colon cancer, which comprises the step of detecting a tannasehigh-producing bacterium contained in an intracolonic microflora sample.4. A test method for colon cancer, which comprises the step of detectingStaphylococcus lugdunensis contained in an intracolonic microflorasample.
 5. A diagnostic agent for colon cancer, which comprises areagent for measuring an amount of tannase contained in an intracolonicmicroflora sample.
 6. The diagnostic agent according to claim 5, whereinthe amount of tannase is measured based on an enzymatic activity.
 7. Thediagnostic agent according to claim 5, wherein the amount of tannase ismeasured by an immunoassay.
 8. A test method for colon cancer, whichcomprise the step of measuring an amount of tannase contained in anintracolonic microflora sample.
 9. The method according to claim 8,wherein the amount of tannase is measured based on an enzymaticactivity.
 10. The method according to claim 8, wherein the amount oftannase is measured by an immunoassay.